Figure 4.

Observation of the pUC19 plasmid DNA states during cleavage by Spd1. Aliquots were taken from the reaction mixture at the indicated time points, and digested products were analysed by electrophoresis in 1% agarose gel, staining with SYBR® Safe (Invitrogen). Control lanes 1: Hyperladder I (Bioline); Lane 2: Untreated pUC19; Lane 3: EcoRI linearized pUC19; Lane 4: Reaction mixture prior to the addition of Spd1. The digestion of plasmid DNA proceeds via single-strand nick mediated relaxation of supercoiled DNA, this can be seen 30 s to 1 min into the reaction; within the same time frame a linear form can also be seen emerging.